Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
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The design and selection of therapeutic molecules having a high degree of target specificity is a major goal of pharmaco-mimetic research. With the rapid development of complex computational algorithms, in silico screening is now a reasonable approach to rational drug design. The difficulty, however, is the identification of not only target molecules but conformationally active pockets, clefts and sites on these molecules.
The identification of therapeutic molecules useful in inflammation is particularly important.
Inflammation is a complex multifactorial process, which includes the migration of neutrophils and monocytes from blood into tissue at inflammatory sites. This migration involves a series of sequential steps proceeding from tethering on endothelium under shear conditions in postcapillary venules (Smith, Microcirculation 7:385-394, 2000). The tethering event depends on adhesion molecules in the selectin family, E-selectin and P-selectin on the endothelium and L-selectin on the neutrophil as well as their respective ligands expressed on both cell types (Burns et al, Physiol Rev 83:309-336, 2003). The adhesion step primarily involves the interaction of integrins (αLβ2, αMβ2, α4β7, α4β1) with adhesion molecules of the Ig-superfamily (ICAM-1, ICAM-2, MadCAM and VCAM) (Fabbri et al, Inflamm Res 48:239-246, 1999). Whereas, the transendothelial cell migration step involves molecules expressed at the junctions between adjacent endothelial cells.
Chronic inflammatory diseases affecting the lung such as bronchial asthma, chronic obstructive pulmonary disease (COPD) and allergic rhinitis are particularly problematic which cause high levels of morbidity and mortality. Asthma is particularly prevalent and is often triggered by exposure to environmental stimuli such as allergens, pharmacological agents, infectious agents, airborne pollutants and irritants.
One cytokine associated with inflammatory lung conditions is interleukin-13 (IL-13). IL-13 is a 17 kDa glycoprotein which has been cloned from activated T-cells (Zurawski and dVries, Immunol Today 15:19-26, 1994). It is a member of the cytokine family characterized by a tertiary structure of four α-helical bundles. The helices are defined as helix A through D. The turns in the helices are referred to as loops, i.e. AB, BC and CD loops. Other members of this family include IL-2, IL-3, IL-4, IL-5 and GM-CSF. It was originally described as a T cell-derived cytokine that inhibits inflammatory cytokine production, however numerous other functions are also attributable to IL-13. These include the regulation of gastrointestinal parasite expulsion, airway hyperresponsiveness (AHR), allergic inflammation, atopic dermatitis, chronic obstructive pulmonary disease (COPD), tissue eosinophilia, mastocytosis, IgE antibody production, goblet cell hyperplasia, tumor cell growth, intracellular parasitism, tissue remodeling and fibrosis (Wynn, Annu. Rev. Immunol. 21:425-56, 2003). The gene encoding human IL-13 is located on chromosome 5Q31, in the same 3000 kb cluster of genes that encodes IL-3, IL-4, IL-5, IL-9 and GM-CSF. The IL-13 gene is 12 kb upstream from the gene encoding IL-4 and lies in the same orientation, indicating that these genes arose by gene duplication during evolution (de Waal Malefyt and de Vries, The Cytokine Handbook, 3rd Edn. A. W. Thomson, Editor: 427-442, 1998). The IL-13 protein has only 25% homology with IL-4 but it does display some similarities of function due to the sharing of a receptor complex with IL-4.
The IL-13 receptor complex includes the IL-4 receptor (IL-4Rα) chain and two other IL-13 binding proteins, IL-13Rα1 and IL-13Rα2. Both IL-13Rα1 and IL-13 Rα2 bind IL-13 but only IL-13Rα1 interacts with IL-4 despite IL-13Rα2 sharing a 37% homology with IL-13Rα1 (Andrews et al, J. Allergy Clin. Immunol 120:91-97, 2007). IL-13Rα1 by itself binds IL-13 with low-moderate affinity (2-10 nM), but in the presence of the IL-4Rα chain, it binds IL-13 with high affinity (kd ˜300-400 μM). In contrast, IL-13 Rα2 binds IL-13 with high affinity (0.5-1.2 nM) affinity but it appears not to contribute to IL-13 signaling. It has been suggested that it acts as a decoy receptor. Thus, the heterodimeric complex formed by the IL-13Rα1 and IL-4Rα chains constitutes the functional IL-13 receptor (Wills-Karp, Immunol. Rev. 202:175-190, 2004). There is a sequential binding sequence for this receptor complex where IL-13 first binds IL-13Rα1 and then recruits IL-4Rα to form a high affinity binding site (Andrews et al, J. Immunol. 176:7456-7461, 2006). Heterodimerization of IL-13R causes activation of Janus kinases, TYK2 and JAK1, constitutively associated with IL-13Rα1 and IL-4Rα, respectively, followed by activation of the signal transducer and activator of transcription 6 (STAT6).
The IL-13R (IL-4Rα/IL-13Rα1) is expressed on hemopoietic and non-hemopoietic cells including B cells, monocytes/macrophages, dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells, airway epithelial cells and airway smooth muscle cells. IL-13 Rα2 has been found on airway smooth muscle cells and airway epithelial cells. IL-13 Rα2 has been described as a decoy receptor and a fusion protein consisting of IL-13 Rα2 and the Fc portion of immunoglobulin is used as an inhibitor of IL-13 in vitro and in vivo. IL-4 can also use IL-13R (IL-4Rα/IL-13 Rα1) as well as another receptor consisting of IL-4Rα and the common γ-chain of the IL-2 receptor. This overlap of receptors accounts for many of the functional similarities of IL-4 and IL-13 (Andrews et al, 2006 supra).
The sites on the IL-13 molecule recognized by each of the receptor chains differ. For example molecular modeling has suggested that the D-helix of IL-13 interacts with IL-13Rα1 whereas parts of the A and C-helices of IL-13 interact with the IL-4Rα chain of the IL-13R (Oshima and Puri, J. Biol. Chem. 276:15195-15191, 2001; Zuegg et al, Immunol. Cell Biol. 79:332-339, 2001). Mutations of glutamic acids at positions 12 and 15 in helix A and arginine and serine at positions 65 and 68 respectively in the C helix were found to be important for biological signaling through the IL-4Rα chain since their specific mutation resulted in loss and/or gain of function (Thompson and Debinski, J. Biol. Chem. 274:29944-29950, 1999). Indeed, mutation E12K produced a powerful antagonist that inhibits the activities of human IL-13 (Oshima and Puri, 2001 supra). Whereas amino acids in the D-helix have been described as important for binding to IL-13Rα1 and/or IL-13Rα2, these are H102, K104, K105, R108, E109 and R111 (Arima et al, J. Biol. Chem. 280:24915-24922, 2005; Madhankumar et al, J. Biol. Chem. 277:43194-43205, 2002). These data are supported by molecular modeling studies and the crystal structures of the signaling complex of IL-4Rα/IL-13/IL-13Rα1. Analyses of the crystal structure further suggest K104 and R108 on IL-13 are critically important for the interactions with IL-13Rα1 domain 3 (LaPorte et al, Cell 132:259-272, 2008). A stripe of amino acids on the A and D helices demarcates a hydrophobic canyon lined by the alkyl moieties of these amino acids. These side chains form clefts into which the receptors insert to contact the main chains of the cytokine A and D helices and of particular importance are the side chains of amino acids R108 and K104 on the D helix. Whereas it appears that R111 is important for binding to the soluble receptor IL-13Rα2 (Andrews et al, J. Allergy Clin. Immunol. 120:91-97, 2007). Domain 1 of IL-13Rα1 interacts with a hydrophobic saucer-shaped patch formed by the alkyl side chains of M33, D87, K89, T35 (LaPorte et al, 2008 supra).
The ability to modify IL-13 function would aid in the development of medicaments useful in the treatment of inflammatory conditions of the lung including asthma, anaphylaxis, emphysema and COPD, and other diseases to which IL-13 contributes including atopic dermatitis, fibrosis and various cancers, for example B chronic lymphocytic leukemia (B-CLL), Hodgkin's disease, where tumor growth/protection from apoptosis is promoted by IL-13, and other cancers in which IL-13 appears to antagonize tumor immunosurveillance (Wynn, 2003 supra). The link between IL-13 and fibrosis suggests that IL-13 antagonists may also be effective in a variety of situations where chronic exposure to IL-13 triggers excessive healing, tissue remodeling, or the formation of destructive tissue pathology in situations like idiopathic pulmonary fibrosis, chronic graft rejection, bleomycin-induced pulmonary fibrosis, progressive systemic sclerosis, radiation-induced pulmonary fibrosis, hepatic fibrosis and acute respiratory distress syndrome (ARDS).
Particular glycosaminoglycan (GAG) sequences can bind specifically and make unique interactions with a number of biomolecules including chemokines, growth factors, cytokines, proteins of the coagulation cascade (e.g. anti-thrombin Ill (AT-Ill)-pentasaccharide complex) and adhesion molecules. Very few GAG fragments, however, have been developed for therapeutic use, mostly because the synthesis of saccharide blocks is chemically challenging. The antithrombin (AT)-binding pentasaccharide ARIXTRA (Registered trade mark) [Sanofi] has been approved for use in thromboprophylaxis following orthopedic surgery and the GAG mimetic PI-88 (Progen) has progressed to clinical trials as an anti-cancer treatment. Other polyanionic polysaccharides (for example pentosan polysulfate) can also bind biomolecules including chemokines, growth factors, cytokines, proteins of the coagulation cascade (e.g. anti-thrombin III (AT-III)-pentasaccharide complex) and adhesion molecules in a manner resembling that of GAGs or GAG sequences.
There is a need to delineate the nature of the interactions of IL-13 with GAGs. This will enable development of small molecule selective modulators of GAG-IL-13 interactions. This includes blocking IL-13 signaling via its receptor (IL-13R) to thereby treat disease conditions that arise because of this signaling event.